Antibacterial peptide from normal rat serum. 1. Isolation from whole serum, activity, and microbicidal spectrum.
A procedure is described for purification of the primary bactericidal component of normal rabbit serum active in vitro against Bacillus subtilis. A 65 000-fold increase in specific bactericidal activity per milligram of serum protein was obtained, yielding a low molecular weight, heat-stable polypeptide fraction (PC-III) exhibiting biological activity at protein concentrations below 10 ng/mL. This preparation appeared homogeneous as judged by column chromatography and analytical NaDodSO4-polyacrylamide gel eletrophoresis; recovery of serum bactericidal activity was routinely greater than 80%. Analysis of dansylated or 125I-labeled samples in peptide-resolving polyacrylamide gels revealed a single band with an Mr of 1800. Optimal antibacterial activity of PC-III against B. subtilis occurred at an ionic strength of 0.24 and was absolutely dependent upon divalent cations; calcium was the most effective. Under optimum conditions, 4 ng/mL of PC-III reduced the viability of B. subtilis test innocula by 90% within 10 min at 37 degrees C. Listeria monocytogenes, Escherichia coli, and Salmonella typhimurium were all sensitive to the action of PC-III, but higher bactericide concentrations were required to produce similar reductions in viability as observed with B. subtilis. All strains were killed by PC-III concentrations well below 1 microgram/mL, roughly that found in normal serum. The activity of PC-III preparations was significantly reduced by pretreatment with trypsin or proteinase K but not by neuraminidase or periodate.
D000818:Animals; D000900:Anti-Bacterial Agents; D001412:Bacillus subtilis / Q000187:drug effects; D001419:Bacteria / Q000187:drug effects*; D002413:Cations, Divalent; D004353:Drug Evaluation, Preclinical; D007700:Kinetics; D009994:Osmolar Concentration; D010455:Peptides / Q000097:blood* / Q000302:isolation & purification / Q000494:pharmacology; D011817:Rabbits
D000900:Anti-Bacterial Agents; D002413:Cations, Divalent; D010455:Peptides